Crystal violet stain

For the biofilm formation inhibition experiment, the procedures were as previously described (28). Simply put, a bacterial suspension with a concentration of 1.5 × 10⁶ CFU/mL was added to a 96-well plate in a volume of 100 µL, followed by the addition of drugs at concentrations corresponding to 0.5 × FICI for each strain. After co-incubation for 24 hours, the planktonic bacteria were discarded, and the wells were washed twice with PBS. Subsequently, the biofilm was air-dried and fixed. Staining and destaining were performed using 1% crystal violet solution and ethanol, respectively. Finally, the absorbance at 595 nm for each well was measured using a microplate reader (BioTek, Synergy).

For the mature biofilm eradication experiment, the procedures were similar to the biofilm formation inhibition experiment, with the only difference being the cultivation of mature biofilms before adding the drugs, as previously described (29). The bacterial suspension (1.5 × 10⁶ CFU/mL, 200 µL) was added to a 96-well plate, and mature biofilms were formed by direct incubation at 37°C for 24 hours. After discarding the liquid in the wells and washing twice with PBS, drug treatments corresponding to 2 × FICI concentrations for each strain were added for 24 hours. Subsequent air-drying, staining, and destaining steps were the same as those in the biofilm formation inhibition experiment.